bacillus subtilis culture characteristics

Gram Positive Bacteria Bacillus subtilis. Bacillus subtilis is a vaccine production host and delivery vehicle with several advantages, such as low production costs, direct administration, safety to human consumption, and the production of spores with adjuvant effects. subtilis Nakamura, Roberts & Cohan 1999, phenotypically similar to Bacillus atrophaeus and distinguishable from that species only by the pigmentation of the latter (brownish-black pigment production on tyrosine agar). Also, the characteristics of the active substance, such as precipitated quality, molecular The present invention provides a novel microorganism, Bacillus subtilis subspecies Krictiensis, and its mutant, Bacillus subtilis subspecies Krictiensis M 18-91; and a novel antifungal complex, KRF-001, produced from said microorganisms. First known as Vibrio subtilis, this bacterium was discovered by Christian Gottfried Ehrenberg in 1835. However, the mechanism that bacteria utilize cellulose, the most abundant substance in the intestines of herbivores, to produce surfactin remains unclear. B. subtilis strain 11685-3 (B. cereus) was … The liquid culture medium used to incubate B. subtilis bacteria had the following components (g∙L −1, deionized water): beef extract 3.0, tryptone 10.0, and NaCl 5.0. (2004) in their study on inactivation of Bacillus anthracis spore using heat and pressure, also proved that spores of B. anthracis are extremely resistant Thus, B. subtilis is able to tolerate many environmental extremes by transitioning into a dormant state as spores, allowing survival under otherwise unfavorable conditions. Bacillus subtilis is a rod-shaped, Gram-positive bacteria that is found in soil and the gut of humans and some types of animals.Bacillus subtilis is commonly included in probiotic supplement formulations. It is able to grow well in oxygenated environments but can adapt to low-to-no oxygen. To investigate the endocellulase characteristics from Bacillus subtilis 1AJ3, and increase its production yield, this paper cloned an endocellulase gene denoted CEL-5A from strain 1AJ3 and expressed in E. coli BL21 (DE3). Some types of Bacillus bacteria are harmful to humans, plants, or other organisms. Bacillus subtilis J2 bacteria were preserved at −20 °C in the laboratory of Zuozhen Han. Biochemical Test of Bacillus subtilis. Bacillus licheniformis is a bacterium that is commonly found in soil and bird feathers. tified as Bacillus subtilis on the basis of its morphological, biochemical and physiological characteristics, and 16S r DNA sequence. It is one of the best-characterized species of all the Gram-positive bacteria. A Bacillus subtilis bacterial colony entering the log phase of growth after 18–24 hours of incubation at 37 °C (98.6 °F; magnified about 6×). Some strains of Bacillus subtilis Strains TS 01 and ZR 02 exhibited the most pronounced inhibitory effects. This Bacillus subtilis gives us a discovery to investigate why it is more effective than Gram-negative bacteria than other bacteria in managing mastitis pathogen. Casein Hydrolysis Test: Purification of Nattokinase from the Culture Supernatant of Bacillus subtilis: The extracellular nattokinase of Bacillus subtilis was produced.The culture supernatant was obtained by centrifugation at 10,000 rpm for 15 min. al. . The following figures (5, 6, and 7) from the CDC are reliable images of Bacillus anthracis grown as described in the figure legends. For example, B. cereus sometimes causes spoilage in canned foods and food poisoning of short duration. Most Bacillus spp grow readily on nutrient agar or peptone media. Basic Characteristics for Identification of Selected Bacillus Species a. Bacillus cereusis well known as an agent of food poisoning, and a number of other Bacillusspecies, particularly B subtilisand B licheniformis, are also incriminated periodically in this capacity. Bacillus subtilis use as a fungicide fortunately does not affect humans (EMBL EBI). -Polyglutamic Acid Produced by Bacillus subtilis (natto): Structural Characteristics, Chemical Properties and Biological Functionalities ... the structural characteristics and their biological function-alities for further development and more practical applica- ... tion. There are often comma shaped projections from the colony edge producing a “Medusa-head” colony. According to our results, the optimum pH for the growth of Bacillus subtilis (strain WT 168) is 8. d. Bacillus subtilis – optimum temperature The temperature has been studied at 3 different values, and at a constant pH (7). Typical Bacillus spp. However, as the material at our Growth of Bacillus subtilis NRS-762 in growth medium Stock cultures of B. subtilis NRS-762 were prepared in 40% glycerol and kept at -70 oC until use. When the bacteria were inoculated into a thermophilic composting reactor for sewage sludge, they survived and Bacillus licheniformis produces stereochemically various DL-PGAs (D-isomer contents, 10 to 100%) [1,6,15]. Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase-positive bacterium (2). Bacillus subtilis is gram positive, rod shape, motile, spore forming bacteria. They are used on plants as a fungicide. Bacillus subtilis (B. subtilis) is a Gram-positive, aerobic bacterium. Characteristics Like all members of the genus Bacillus, B. subtilis is a rod-shaped bacterium that typically forms small clumps, short chains, or … Bacillus subtilis) to challenge the limits of microbial resistance and survival. Properties (Bacillus subtilis) Catalase. — Colony Characteristics. Biochemical Test and Identification of Bacillus subtilis. As shown in Fig. and 1 -5 microns in length. Bacillus stearothermophilus α/β-cyclodextrin glycosyltransferase (α/β-CGTase) is an excellent transglycosylase with broad potential for food application, but its expression level is low in Bacillus subtilis. L-PGA is produced by an alkalophilic Bacillus [17], although it is quite different from D-PGA and Non-typical Bacillus strains may be very small and spore formation often fails to occur. One of the first bacteria to be studied. 2b, the colonies of ASAG 216 appeared to have irregular edges and a milky white wrinkled surface. Peptidoglycan surrounds the cell membrane and gives the bacterial cell shape and structure.Bacteria Description and significance. Methods and Results: Cultured cell lines, Caco-2, HEp-2 and the mucus-producing HT29-16E cell line, were used to evaluate adhesion, invasion and cytotoxicity. The AIM of the investigation was to characterize the DNA as a part of nucleoprotein fraction isolated from Bacillus subtilis culture medium filtrate (CMF) and to evaluate the dynamic of accumulation of such DNA and level of its unmethylated CpG motifs enrichment in the … ; The optimum temperature for growth varies from 20°C to 40°C, mostly 37°C. Figure 5. REGNUM PROKARYOTAE - Cultural characteristics. Most do not grow well on enteric agars. Let them grow overnight at 37°C, 200 rpm 3. Bacillus Subtilis Morphology Bacillus subtilis morphology describes rod-shaped, Gram-positive bacteria that show up on both positive and negative Gram stain techniques. Microscopic Characteristics: Rod-like or cylindrical in shape, measuring 0.2-1.2 microns in diameter. No significant The strain ASAG 216 is a long, rod-shaped, and Gram-positive bacterium (Fig. L-PGA is produced by an alkalophilic Bacillus [17], although it is quite different from D-PGA and The work describes the production of alkaline protease in a lab scale fermentor from Bacillus subtilis … Birds that tend to stay on the ground more than the air (i.e. γ‐(D,L)‐Polyglutamic acid [γ‐(D,L)‐PGA, or γ‐PGA] produced by Bacillus subtilis (natto) both on laboratory and pilot scale fermenter systems has been characterized. Aims: To conduct in vitro and in vivo assessments of the safety of two species of Bacillus, one of which, Bacillus subtilis, is in current use as a food supplement. Originally named Vibrio subtilis in 1835, this organism was renamed Bacillus subtilis in 1872. A bacterial rod is a symmetrical cylinder with rounded ends. Suppressiveness of the culture broth of the bacteria was investigated using other phytopathogenic fungi in vivo and the broth showed a broad suppressive spectrum. Long chains in liquid culture but may include single cells or short chains . Bacillus subtilis is an aerobic, Gram-positive soil bacterium, which has been widely used for the production of heterologous proteins [].It secretes numerous enzymes to degrade a variety of substrates, enabling the bacterium to survive in a continuously changing environment. Strip Dimensions. Carbon source is an important factor for surfactin production. ­Basic Characteristics. ridges along the margins of individual grain kernels characterize this contaminant. typically forms small clumps, short chains, or single cells. 3 subspecies: - B. subtilis subsp. The different species of Bacillus produce a variety of extracellular products including antimicrobial substances, enzymes, pigments, and toxins in few species ( 42 ). Aspects such as increasing yield using various starter culture volumes, various media and incubation time and temperatures, were studied. Bacillus subtilis is also known as Hay bacillus or Grass bacillus. According to reviews, the optimum temperature growth of Bacillus subtilis is 37°C. Incubation conditions were 30 oC and 230 Bacillus is a genus of bacteria in the Firmicutes division that are characterized by being spore-forming under aerobic conditions. Bacillus spp. ; On Nutrient Agar at 37°C, it forms large (2-5 mm) grey-white, granular colonies with a less wavy edge and less membranous consistency. Bacillus subtilis as Vaccine-vectors. Bacillus subtilis is a typical germ, which is rod-shaped and Gram-positive. Browse 1,356 bacillus subtilis stock photos and images available, or search for escherichia coli or staphylococcus aureus to find more great stock photos and pictures. The Bacillus species B. subtilis, B. licheniformis, and B. pumulis are closely related, and historically, there has been difficulty distinguishing among the three species. They are rod-shaped and are generally Gram positive (although some are Gram variable), with colonies that present organisms that stain pink and others that stain purple. 1000 kDa) are obtained from the culture of B. subtilis (natto). Positive (+ve) Citrate. Bacillus subtilis - solid & liquid media. Colonies of Bacillus cereus on the left; colonies of Bacillus anthracis on the right. Bacillus subtilis is one of the most studied bacteria with very well definied characteristics as its entire genome has been sequenced . It was found that the bacteria are Gram positive spore chain Bacillus. exhibit large, flat colonies on non-selective media. Bacillus megaterium also synthesizes DL-PGA (D-isomer content, 50%) [16]. After optimum time of incubation in optimum temperature, bacterial colony appears on the culture plate. One glycerol stock culture of B. subtilis NRS-762 was used in inoculating 100 mL of LB Lennox medium in a 250 mL glass conical flask as seed culture. The isolated strain, which was found to be capable of producing said antifungal agent, KRF-001, was named Bacillus subtilis subspecies Krictiensis and deposited on Jul. ix 4.2 Growth curve of Escherichia coli and Bacillus subtilis 35 4.3 Colony Forming Unit (CFU) 37 4.5 Effect of exposure time and cell concentration on bacteria adhesion42 4.6 Effect of culture age 46 A.W. Pallid to whitish. Spores are subterminal or central Additional Information . Bacillus subtilis commonly present in soil which is human pathogens. 26, 1990 with American Type Culture Collection under the deposit number of ATCC 55079. Bacillus megaterium also synthesizes DL-PGA (D-isomer content, 50%) [16]. Inoculate a culture of Bacillus subtilis in 4 ml of LB-Medium 2. 7 … Bacillus subtilis Cell Line 5230. To investigate the texture and cooking properties of garaetteok elaborated with potato flour, experimental products were elaborated replacing partially the rice flour, traditionally used, with nonfermented (NF) or fermented potato flour with Bacillus subtilis (BS) or Leuconostoc mesenteroides (LM) in percentages of 15% (NF15, BS15, and LM15) and 20% (NF20, BS20, and LM20). Bacillus subtilis is one of the best characterized bacteria and is used as a model organism for Gram-positive bacteria. Bacillus cereus is a large gram positive, rod shaped bacteria that produce toxins and is capable of producing endospores. The bacterium Bacillus subtilis has long been an important subject for basic studies. Under a very high magnification of 31,207X, this scanning electron micrograph depicted spores from the Sterne strain of Bacillus … It's a useful and beneficial probiotic that supports digestion, enzyme production, immune and digestive system health. Indications for Use. For example, Bacillus species pseudobacteremia has been traced to contaminated gloves used in collection of blood from patients . Bacillus subtilis is a rod-shaped bacterium that secretes numerous enzymes to degrade a variety of substances during metabolism. Bacillus subtilis were inoculated at the center of a dish with gelose containing nutrients. Characteristics of Bacillus subtilis. A member of the genus Bacillus, B. subtilis is rod-shaped, and can form a tough, protective endospore, allowing it to tolerate extreme environmental conditions. A significant difference in pressure across the cytoplasmic membrane pushes the cell wall into a specific shape. The bacteria, colonized on root systems, compete with disease causing fungal organisms. Eugon broth as a culture medium enhanced the germination and reduced the incidence of rust pustules on inoculated leaves antagonistic effect compared with cultures produced in nutrient broth. Bacillus licheniformis produces stereochemically various DL-PGAs (D-isomer contents, 10 to 100%) [1,6,15]. It has a cell wall that is made of a complex molecule called peptidoglycan, which is made of long chains of glucose linked together by amino acids. Cultural morphological and biochemical characteristics of the tested strains conformed to the species characteristics of Bacillus subtilis. However, as the material at our Bacillus subtilis Z-14 was isolated from soil sampled from a wheat rhizosphere and was confirmed to have strong antifungal activity against R. cerealis. The present invention relates to a feed additive for fish containing novel microorganism Bacillus subtilis SJ-30. Structure and Physiology Bacillus subtilis is a spore forming, motile, rod-shaped, Gram-positive, facultative aerobe. Thus, B. subtilis is able to tolerate many environmental extremes by transitioning into a dormant state as spores, allowing survival under otherwise unfavorable conditions. Phylum Firmicutes, Class Bacilli, Order Bacillales, Family Bacillaceae, Genus Bacillus, Bacillus subtilis (Ehrenberg) Cohn 1872 Smith et al.,1946. B. subtilis SPB1 strain was streaked on a nutrient agar slant [17] and incubated at 37°C.After 24 h, one loop of cells was dispensed in 3 mL of LB medium and incubated overnight at 37°C. ducks) are common carriers of this bacterium; it is mostly found around the bird's chest area and back plumage. In the Primary in vitro screening for antagonism of the isolates was performed against five phytopathogenic mould fungi. 2. B. cereus is mesophilic and is capable of adapting to a wide range of environmental conditions. Keywords Bacillus subtilis Bacterial culture optimization Spore production Sporulation efficiency Introduction Aerobic Endospore-Forming Bacteria (AEFB), especially some species of Bacillus spp., have received growing attention because of their industrial potential in the development of biopesticides and biofertilizers. Bacillus subtilis bacterial growth phases After 96 hours at 37 °C (98.6 °F), a Bacillus subtilis bacterial colony shrivels, which indicates that it has entered the death phase (magnified about 9×). SOURCES Produced by pure culture fermentation of a nonpathogenic and nontoxigenic genetically modified strain of Bacillus subtilis containing the xylanase gene derived from B. subtilis. sparrows)and on the water (i.e. Sheep blood agar (SBA) Colonies are nonhemolytic, flat or slightly convex with irregular edges and ground-glass appearance. B. subtilis has the ability to produce and secrete antibiotics. 1000 kDa) are obtained from the culture of B. subtilis (natto). According to reviews, the optimum temperature growth of Bacillus subtilis is 37°C. report by Shoda (2000) on the special characteristics of Bacillus which is high thermal tolerance, rapid growth in liquid culture and ready formation of resistant spores. Other names for this bacteria also include Bacillus uniflagellatus, Bacillus globigii, and Bacillus natto.Bacillus subtilis bacteria were one of the first bacteria to be studied. The culture supernatant was obtained by centrifugation at 10,000 rpm for 15 min. The bacteria start mass-migrating outwards about twelve hours after inoculation, forming dendrites which reach the border of the dish (diameter 90mm) within a few hours. For example, Bacillus species pseudobacteremia has been traced to contaminated gloves used in collection of blood from patients . Although it was nominated to the DSL as a strain of B. subtilis, and is listed in culture collections under that species name, B. subtilis strain 11685-3 was discovered by Health Canada scientists to be a strain of B. cereus. Hi there, When we attempt to identify an unknown bacteria we should always pay proper attention to the isolation of the species at first. makes uncolonized grain appear excessively wet, hence the name “Wet Spot”. Related information on delivery / use of the strain One glycerol stock culture of B. subtilis NRS-762 was used in inoculating 100 mL of LB Lennox medium in a 250 mL glass conical flask as seed culture. “B. When cultured on ordinary nutrient agar, the When cultured on ordinary nutrient agar, the Bacillus subtilis bacteria are non-pathogenic. In immunocompromised hosts, a blood culture growing Bacillus species should be evaluated carefully. 1. Inoculate a culture of Bacillus subtilis in 4 ml of LB-Medium 2. Let them grow overnight at 37°C, 200 rpm 3. Measure the OD 600 of your overnight culture and dilute it in MNGE Medium to an OD 600 of around 0.1-0.2/ml in 10 ml LB 4. Let the cells grow to an OD 600 of 1.0-1.3/ml (37°C, 200 rpm) 5. Cells should be competent now (very motile) 6. Originally named Vibrio subtilis in 1835, this organism was renamed Bacillus subtilis in 1872 (MicroWiki.com). GFG-160 was protoplast-cured (Edger et af., 1981) of pSA33 before use as a host.Plasmid stability Table 3 .3Efect of storage on the stability characteristics of deriued strains of Bacillus subtilis BCIStrains were derived from chemostat culture as described in the text, and were stored at … ... Endospore in Bacillus subtilis; Bacillus spp are aerobic spore forming rods that stain gram positive or gram variable. The results also indicate that bacteriocin produced by the Bacillus subtilis against the test organism show is the best alternative in the management of the mastitis. In addition to being a cell factory for pharmaceutical proteins, B. subtilis has many industrial and environmental applications. They are also used on agricultural seeds, such as vegetable and soybean seeds, as a fungicide. In immunocompromised hosts, a blood culture growing Bacillus species should be evaluated carefully. Measure the OD 600 of your overnight culture and dilute it in MNGE Medium to an OD 600 of around … The ability to form endospores allows certain Gram-positive bacteria (e.g. The Spore Strips may be utilized to monitor Steam sterilization process efficacy. This bacteria is a prime example for cellular development. However, this major cluster … They were identified as Bacillus subtilis on … However, this organism has also had industrial applications due to its easy genetic manipulation, favorable culturing characteristics for large‐scale fermentation, superior capacity for protein secretion, and generally recognized as safe (GRAS) status. Saved by Edie Kyles. Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a Gram-positive, catalase -positive bacterium, found in soil and the gastrointestinal tract of ruminants and humans. as the subtilis-group or subtilis-spectrum (Gordon, 1973). Competent #1 - Chemo-Competent Bacillus subtilis 1. Except for few species the large majority have no Bacillus are often isolated on blood culture and usually represent blood culture contamination. These three species clustered together (78%) in the "subtilis" group in a numerical classification based on 118 unit characteristics of 368 strains of Bacillus (Priest et al., 1988). This is due to the ubiquitous nature of the microorganism in the environment. B. subtilis is often used as a safer substitute for other pathogenic varieties of Bacillus to determine the likely effects on Bacillus species and/or spore-forming microorganisms in general. It has an optimum growth temperature in the range of 30-39°C. Endocellulase is a key cellulase for cellulosic material pretreatment in the industry by hydrolyzing long cellulose chains into short chains. The ability to form endospores allows certain Gram-positive bacteria (e.g. subtilis, isolated from a rust-infected geranium leaf, inhibited spore with a culture filtrate. Bacillus subtilis: Soil Organism or Probiotic?Or Both? They are often beta-hemolytic. Source: S. Zamenhof's strain 31 (trpC2 hisB31) transformed with DNA of Bacillus subtilis 15 (trpB3 arg15) by H. Saito (originally derived from Bacillus subtilis Marburg strain). Positive (+ve) Flagella. Biosafety Level 2 agent (Use BSL3 for large volume or high titer culture) . 1d). Cléry-Barraud et al. It was renamed in 1872 by Ferdinand Cohn. The application of CpG DNA is one of the most modern and sufficiently perspective trends in immunotherapy of cancer. Bacillus subtilis) to challenge the limits of microbial resistance and survival. Characteristics of Bacillus subtilis isolated from composts suppressing phytopathogenic ... Bacteria which were associated with the effect were isolated and identified as Bacillus subtilis. Yongsoo Park, Hiroyuki Uehara, Ryou Teruya, Mitsuyasu Okabe, Effect of culture temperature and dissolved oxygen concentration on expression of α-amylase gene in batch culture of spore-forming host, Bacillus subtilis 1A289, Journal of Fermentation and Bioengineering, 10.1016/S0922-338X(97)82786-9, 84, 1, (53-58), (1997). It’s capability of controlling Fusarium wilt in green house and in the field was evaluated (Liu et. Rakosy/Encyclopædia Britannica, Inc. They can contaminate food, however, they seldom result in food poisoning. Bacillus subtilis means thin and rod-shaped and it was first discovered in the 1870’s. It is mostly found in soil and vegetation with an optimal growth temperature from 25-35 degrees Celsius. Bacillus subtilis belongs to group II, whose distinguishing features are (i) the formation of an ellipsoidal spore which does not swell the mother cell, (ii) the ability of cells to grow anaerobically with glucose as the carbon source in the presence of nitrate, and (iii) the production of … Antagonistic Bacillus strains were isolated from soil and analyzed for the purpose of determining whether they could be used as natural biological agents. S = isolates from soil, FS = isolate from fermented soybean. Surfactin secreted by Bacillus subtilis can confer strong, diverse antipathogenic effects, thereby benefitting the host. MIcroscopie à contraste de phase (400X) et microscopie standard (1000X). Can be misidentified as: Bacillus megaterium and other . Colonies have a sticky consistency when manipulated with a loop. Characteristics Of Bacillus Subtilis. The genus Bacillus consists of gram-positive, endospore-forming, rod-shaped bacteria. There are more than 70 species, which display wide morphological and physiological diversity. Only 2 (B. anthracis and B. cereus) are known to be human pathogens. Bacillus anthracis. 2011). Studies show that there is a high genetic variety in B. subtilis even in strains harvested from the same source. Some strains are capable of producing toxins. Some products have listed “B. subtilis” on their labels when in fact they were another Bacillus species known to produce toxins such as Bacillus cereus. Bacillus subtilis is typically utilized to monitor low temperature steam processes (<121°C). The biochemical and physiological characteristics of the strain ASAG 216 indicated that it belongs to the genus Bacillus (Table 1).A comparison of the 16S rDNA gene sequence of the isolate … Gordon (1973), who conducted much of the pioneering work on the taxonomy of the genus, referred to these three species as the subtilis-group or subtilis-spectrum. Phenotypic characteristics and Phenotypic changes ... Bacteria are cultured in artificial culture media provided with all the minimal nutrients required for growth and multiplication. Enzymes that can be found on culture include amylase, collagenase, hemolysin, lecithinase, phospholipase, protease, and urease. Bacillus are often isolated on blood culture and usually represent blood culture contamination. Tested strains were nonpathogenic for white mice. According to our results, the optimum pH for the growth of Bacillus subtilis (strain WT 168) is 8. d. Bacillus subtilis – optimum temperature The temperature has been studied at 3 different values, and at a constant pH (7). The underlying mechanisms of pulp modification and different effects on various types of pulps by the EG treatment were also discussed by studying the change in fibers characteristics … In LB broth medium, surface biofilm generation, fluid and transparent culture, bottom floc after the shock, indicate that the bacteria are aerobic, consistent with the characteristics of Bacillus subtilis. γ‐PGA in its free acid form (H +) is insoluble in water.The salt forms of K +, Na +, NH 4 +, Ca 2+, and Mg 2+ of γ‐polyglutamates are fully soluble in water. Wheat sheath blight, a soil borne fungal disease caused by Rhizoctonia cerealis, is considered as one of the most serious threats to wheat worldwide. identified as Bacillus subtilis. Through isolation and culture of the unknown bacteria, the culture characteristics, morphology observation, biochemical test, preliminary antibacterial test, 16S rRNA PCR amplification, sequence analysis, and homology analysis were performed. A member of the genus Bacillus, B. subtilis is rod-shaped, and has the ability to form a tough, protective endospore, allowing the organism to … B. subtilis is a rod-shaped bacterium, which produces endospores that allow the survival of extreme environmental conditions including heat and desiccation. B. subtilis bacteria are rod shaped, Gram positive bacteria that are generally found within the soil and plants. Incubation conditions were 30 oC and 230 The feed additive for fish of the present invention exhibits an antibacterial effect against fish pathogenic bacteria (Streptococcus parauberis), and contains, as an active ingredient, a novel microorganism Bacillus subtilis SJ-30 strain, KCTC18249P. It is rod-shaped and catalase-positive. Bacillus subtilis biochemical tests. cereus is mesophilic, growing optimally at temperatures between 20°C and 40°C, and is capable of adapting to a wide range of environmental conditions”, as stated in Bacillus cereus article. Growth of Bacillus subtilis NRS-762 in growth medium Stock cultures of B. subtilis NRS-762 were prepared in 40% glycerol and kept at -70 oC until use.

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